20 Questions
What is the purpose of this SOP?
To describe the selection and use of positive and negative controls
True or false: Positive controls are performed on sections of tissue known to contain the target antigen.
True
What is the purpose of a positive control in immunohistochemistry staining?
Positive controls assess the performance of the primary antibody. They are performed on sections of tissue known to contain the target antigen, using the same immunohistochemistry staining protocol as the patient tissue.
Positive controls assess the performance of the primary antibody.
What is the IHC test label used for?
Varying reagent and instrument testing
False positive staining can be caused by endogenous biotin.
True or false: The UltraView Universal DAB detection kit is intended for the specific and sensitive detection of mouse and rabbit primary antibodies.
True
What is the purpose of a negative control in immunohistochemistry staining?
Negative controls must assess the presence of non-specific staining in patient tissue as well as the specificity of each antibody. An internal negative control can be found within the patient tissue sample.
Internal negative controls can be found within the patient tissue sample.
True or false: A "true positive" stain shows chromogen deposition in cells or structures that truly contain the antigen of interest.
True
What are the components of the IHC test label (AP-HIS.A504)?
The IHC test label (AP-HIS.A504) will be used for varying reagent and instrument testing, utilizing the Ventana Benchmark Ultra Automated Stainer. It includes reagent name, lot number, open date, expiration date, and storage temperature.
What is the UltraView Universal DAB Detection Kit used for?
Detecting mouse and rabbit primary antibodies
What is the ISH iVIEWBlue Plus Detection Kit used for?
Detecting specific DNP labeled probes
True or false: The IHC test label (AP-HIS.A504) will be used for varying reagent and instrument testing.
True
The UltraView Universal DAB detection kit is intended for the specific and sensitive detection of ______ primary antibodies.
Mouse and Rabbit
What is the difference between a true positive and false positive stain?
A "true positive" stain shows chromogen deposition in cells or structures that truly contain the antigen of interest. In contrast, a "false positive" stain is one where the chromogen is localized to cells or structures that in reality lack the antigen of interest.
The ISH Blue plus Detection uses a positive ______ Control (U6) and a negative reagent control.
RNA
What are the two detection kits used for different types of immunohistochemistry staining?
The UltraView Universal DAB detection kit is a multimer-technology based detection system intended for the specific and sensitive detection of mouse and rabbit primary antibodies. The ISH iVIEWBlue Plus Detection Kit detects specific DNP labeled probes and antibodies bound to a target sequence or antigen in paraffin-embedded tissue sections.
True or false: Internal positive patient controls are used in rare cases if the positive control has lost antigenicity or the control did not adhere well to the slide.
True
What is the difference between a true positive stain and a false positive stain?
A true positive stain shows chromogen deposition in cells or structures that contain the antigen of interest, while a false positive stain shows chromogen deposition in cells or structures that lack the antigen of interest.
Study Notes
- The purpose of this standard operating procedure (SOP) is to describe the selection and use of positive and negative controls, as well as false positive staining.
- Positive controls assess the performance of the primary antibody. They are performed on sections of tissue known to contain the target antigen, using the same immunohistochemistry staining protocol as the patient tissue.
- Some cells or tissue contain endogenous biotin which can cause non-specific staining or false positive staining.
- Negative controls must assess the presence of non-specific staining in patient tissue as well as the specificity of each antibody.
- An internal negative control can be found within the patient tissue sample.
- All instrument preventative maintenance function checks and quality control and reagent quality control are performed and documented according to specific standard operating procedures.
- The IHC test label (AP-HIS.A504) will be used for varying reagent and instrument testing, utilizing the Ventana Benchmark Ultra Automated Stainer.
- Non-specific background staining is non-immunological binding of the specific immune sera by hydrophobic and electrostatic forces to certain sites within the tissue.
- Positive controls are designated for each antibody located with the data sheet and in the tissue control block accountability binder in IHC. Internal negative controls can be found within the patient tissue if the pathologist does not designate one.
- Opening a new prep kit uses the reagent information listed below to make the label for the prep kit: Reagent name, lot number, open date, expiration date, and storage temperature.
- The Ventana Benchmark Ultra Automated Stainer can be used to perform lot to lot testing for IHC.
- After the prep kit is filled, it must be QCd by the technician. The IHC test label (AP-HIS.A504) can be used to document the results of the lot to lot testing.
- The HIS staining procedure includes verification of slides by the pathologist or designee, and recording of positive and negative controls.
- The HIS procedure is approved and current, effective starting 30-Jun-2022.
- BAMC maintains a bank of formalin-fixed controls and some commercial controls for rare antigens and detection lot testing.
- Internal positive patient controls are used in rare cases if the positive control has lost antigenicity or the control did not adhere well to the slide.
- The UltraView Universal DAB detection kit is a multimer-technology based detection system intended for the specific and sensitive detection of mouse and rabbit primary antibodies.
- The UltraView Universal Red detection kit is a multimer-technology based detection system intended for the specific and sensitive detection of mouse and rabbit primary antibodies.
- The Ventana's OptiView DAB IHC Detection Kit is an indirect, biotin-free system for detecting mouse IgG, mouse IgM and rabbit primary antibodies.
- The ISH iVIEWBlue Plus Detection Kit detects specific DNP labeled probes and antibodies bound to a target sequence or antigen in paraffin-embedded tissue sections.
- The labeled probe or antibody is located by an anti-DNP antibody, then by an enzyme-labeled secondary antibody or a biotin-conjugated secondary antibody. This step is followed by the addition of a Streptavidin-AP (alkaline phosphatase) enzyme conjugate which binds to the biotin present on the secondary antibody. The complex is then visualized with 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) chromogen, which produces a blue precipitate that is readily detected by light microscopy.
- A "true positive" stain shows chromogen deposition in cells or structures that truly contain the antigen of interest. In contrast, a "false positive" stain is one where the chromogen is localized to cells or structures that in reality lack the antigen of interest.
- The ISH Blue plus Detection uses a positive RNA Control (U6) and a negative reagent control.
- The Optiview DAB Detection uses a negative mouse and negative Current.
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